Pigs, especially piglets, suffer from scours, that is, diarrhea, which can be caused by bacteria such as Escherichia coli (E. coli ) and Clostridium perfringens Types A and C (C. perfringens). Scours can cause death losses and severe production losses, including weight loss, if left untreated.
E. coli is the primary cause for diarrhea in piglets and 50-75% of the antibiotic used on farms is used against weaning diarrhea, primarily caused by E. coli. Diarrhea is the biggest problem in weaners and growers (up to 40 kg) and E. coli is the most important pathogen causing diarrhea (Klose et al., 2010).
Enteric clostridial infections in swine occur predominantly in the preweaning period but are also associated with hemorrhagic bowel syndrome affecting pigs in the finishing period. Although immunization against C. perfringens type C has greatly reduced pre-weaning mortality, no commercial vaccines are currently available for C. perfringens type A. C. perfringens type A infections are now recognized with increasing frequency in preweaning pigs and approaches to diagnosis and prophylaxis are both different and more complex than those for type C infections.
Several infections and diseases in poultry are caused by pathogenic bacteria, including E. coli and Clostridium perfringens. Infections and diseases caused by pathogens result in increased mortality, decreased performance, and increased production costs. In addition, many of these pathogens can be transmitted to humans. Avian colibacillosis is a systemic infection caused by E. coli and occurs most commonly in young broilers and poults.
Probiotics are used in animal health applications in order to maintain healthy gut microflora, including a reduction in detrimental bacteria such as Clostridia and E. coli and an increase in beneficial bacteria such as Lactobacillus spp. and Bifidobacterium. Probiotics are well-suited to maintaining a healthy balance between pathogenic and beneficial bacteria because, unlike antibiotics, they do not destroy bacteria indiscriminately nor do they lead to antibiotic resistant strains of pathogenic bacteria. There are many mechanisms by which probiotics are thought to maintain healthy gut microflora: competitive exclusion of pathogenic bacteria, reduction of pathogenic bacteria through production of antimicrobial substances, enhancing growth and viability of beneficial gut microflora, and stimulating a systemic immune response in the animal.
In view of the foregoing, it would be desirable to have one or more Bacillus strains to treat or prevent diseases due to infections with E. coli and/or Clostridium in pigs and poultry.
Guo et al., 2006, describes screening of Bacillus strains as potential probiotics and a test of Bacillus subtilis MA139 in pigs. 124 samples were collected from broiler, pigs, soils, fermented foods and Chinese herbs. 750 aerobic spore-forming strains were isolated from these samples.
Inhibitory activity against E. coli K88 and K99, Salmonella and Staphylococcus aureus was tested using a disc plate diffusion assay. 6 Bacilli with best activity were tested for their survival within simulated GIT conditions (pH 2 and 0.3% bile salt). B. subtilis MA139 was the best candidate and was tested in vivo in piglets in a 28 days feeding trial with 90 piglets. ADG and feed utilization was improved. Lactic acid bacteria were increased, E. coli in feces was decreased. However, antimicrobial activity against Clostridium perfringens and sensitivity to antibiotics were not tested.
Barbosa et al., 2005 describes isolation of 237 Bacillus from feces from organically (contact to soil) reared broilers. 31 isolates were characterized. B. subtilis and B. licheniformis were among these. Several B. subtilis strains showed inhibition to C. perfringens and S. aureus. B. licheniformis also showed effect against C. perfringens. However, none of the selected Bacillus isolates exhibited antimicrobial activity against E. coli as defined in the present application. One selected Bacillus isolate shows reduction in growth intensity but not complete inhibition against E. coli strain O78:K80 and no effect against the other E coli strain tested (see Table 5). No data is provided on the sporulation percentage after 2 days of incubation or on the sensitivity to vancomycin, kanamycin, streptomycin, and clindamycin.
Chaiyawan et al., 2010, discloses a Bacillus strain sp. T3-1, which is susceptible to antibiotics widely used in medical treatment and which shows antimicrobial activity against C. perfringens ATCC 15191. The strain has no antimicrobial activity against E. coli O157. No data on the sporulation percentage after 2 days of incubation provided.
Benitez et al., 2011 has recently described that the presence of intact or inactivated E. coli enhanced the synthesis of antimicrobial peptides by Bacillus amyloliquefaciens LBM 5006 strain.
U.S. Pat. No. 7,754,469 relates to microorganisms and methods for treating poultry and U.S. Pat. No. 8,021,654 relates to methods of treating pigs with Bacillus strains.
However, in none of these articles or patents there is any description or suggestion to select for Bacillus strains that are sensitive for antibiotics which are commonly used for humans, have antimicrobial activity against both Clostridium perfringens and E. coli and have a high sporulation percentage in order to make the strain useful for efficient sporulation and thus Bacillus probiotic production.
None of the prior art documents e.g. Barbosa et al., 2005, Chaiyawan et al., 2010, and Guo et al., 2006 disclose strains having sensitivity for antibiotics which are commonly used for humans, antimicrobial activity in the sense of inhibition of growth against both Clostridium perfringens and E. coli, and a high sporulation percentage.
In summary, the prior art relating to screening of Bacillus strains does not provide the three distinguishing features of the present invention, i.e. sensitivity for antibiotics which are commonly used for humans, antimicrobial activity against E. coli and Clostridium perfringens and a high sporulation percentage. Nor does the prior art provide Bacillus strains fulfilling these three criteria.